1H-1,2,3-triazol-1,4-naphthoquinone Derivatives: Novel Inhibitors Targeting Pyocyanin Biosynthesis for P. Aeruginosa Infection Treatment Advances.

BACKGROUND: This study investigates the potential of eleven 1H-1,2,3-triazol-1,4-naphthoquinone conjugates as virulence factor inhibitors (like Pyocyanin) and their affinity for PhzM, a crucial enzyme for Pyocyanin biosynthesis in Pseudomonas aeruginosa infections. METHODS: A straightforward synthetic pathway enabled the production of these compounds, which were characterized and structurally confirmed through spectroscopic analyses. Evaluation of their impact on PhzM thermal stability identified promising candidates for PhzM binders. RESULTS: Concentration-response behavior elucidated their binding affinity, revealing them as the first reported micromolar affinity ligands for PhzM. Structure-activity relationship analysis emphasized the role of specific molecular moieties in binding affinity modulation, paving the way for future advanced inhibitors' development. CONCLUSION: These findings highlight the potential of naphthoquinone-triazole derivatives as leads for novel therapeutics against P. aeruginosa infections.

Pseudomonas aeruginosa strains belonging to phylogroup 3 frequently exhibit an atypical quorum sensing response: the case of MAZ105, a tomato rhizosphere isolate.

Pseudomonas aeruginosa is a widespread γ-proteobacterium and an important opportunistic pathogen. The genetically diverse P. aeruginosa phylogroup 3 strains are characterized by producing the pore-forming ExlA toxin and by their lack of a type III secretion system. However, like all strains of this species, they produce several virulence-associated traits, such as elastase, rhamnolipids and pyocyanin, which are regulated by quorum sensing (QS). The P. aeruginosa QS response comprises three systems (Las, Rhl and Pqs, respectively) that hierarchically regulate these virulence factors. The Pqs QS system is composed of the PqsR transcriptional factor, which, coupled with the alkyl-quinolones HHQ or PQS, activates the transcription of the pqsABCDE operon. The products of the first four genes of this operon produce HHQ, which is then converted to PQS by PqsH, while PqsE forms a complex with RhlR and stabilizes it. In this study we report that mutations affecting the Pqs system are particularly common in phylogroup 3 strains. To better understand QS in phylogroup 3 strains we studied strain MAZ105 isolated from tomato rhizosphere and showed that it contains mutations in the central QS transcriptional regulator, LasR, and in the gene encoding the PqsA enzyme involved in the synthesis of PQS. However, it can still produce QS-regulated virulence factors and is virulent in Galleria mellonella and mildly pathogenic in the mouse abscess/necrosis model; our results show that this may be due to the expression of pqsE from a different PqsR-independent promoter than the pqsA promoter. Our results indicate that using anti-virulence therapy based on targeting the PQS system will not be effective against infections by P. aeruginosa phylogroup 3 strains.

To cheat or not to cheat: cheatable and non-cheatable virulence factors in Pseudomonas aeruginosa.

Important bacterial pathogens such as Pseudomonas aeruginosa produce several exoproducts such as siderophores, degradative enzymes, biosurfactants, and exopolysaccharides that are used extracellularly, benefiting all members of the population, hence being public goods. Since the production of public goods is a cooperative trait, it is in principle susceptible to cheating by individuals in the population who do not invest in their production, but use their benefits, hence increasing their fitness at the expense of the cooperators' fitness. Among the most studied virulence factors susceptible to cheating are siderophores and exoproteases, with several studies in vitro and some in animal infection models. In addition to these two well-known examples, cheating with other virulence factors such as exopolysaccharides, biosurfactants, eDNA production, secretion systems, and biofilm formation has also been studied. In this review, we discuss the evidence of the susceptibility of each of those virulence factors to cheating, as well as the mechanisms that counteract this behavior and the possible consequences for bacterial virulence.

N-acetylcysteine (NAC) attenuates quorum sensing regulated phenotypes in Pseudomonas aeruginosa PAO1.

The expression of many virulence genes in bacteria is regulated by quorum sensing (QS), and the inhibition of this mechanism has been intensely investigated. N-acetylcysteine (NAC) has good antibacterial activity and is able to interfere with biofilm-related respiratory infections, but little is known whether this compound has an effect on bacterial QS communication. This work aimed to evaluate the potential of NAC as a QS inhibitor (QSI) in Pseudomonas aeruginosa PAO1 through in silico and in vitro analyses, as well as in combination with the antibiotic tobramycin. Initially, a molecular docking analysis was performed between the QS regulatory proteins, LasR and RhlR, of P. aeruginosa with NAC, 3-oxo-C12-HSL, C4-HSL, and furanone C30. The NAC sub-inhibitory concentration was determined by growth curves. Then, we performed in vitro tests using the QS reporter strains P. aeruginosa lasB-gfp and rhlA-gfp, as well as the expression of QS-related phenotypes. Finally, the synergistic effect of NAC with the antibiotic tobramycin was calculated by fractional inhibitory concentrations index (FICi) and investigated against bacterial growth, pigment production, and biofilm formation. In the molecular docking study, NAC bound to LasR and RhlR proteins in a similar manner to the AHL cognate, suggesting that it may be able to bind to QS receptor proteins in vivo. In the biosensor assay, the GFP signal was turned down in the presence of NAC at 1000, 500, 250, and 125 µM for lasB-gfp and rhlA-gfp (p < 0.05), suggesting a QS inhibitory effect. Pyocyanin and rhamnolipids decreased (p < 0.05) up to 34 and 37%, respectively, in the presence of NAC at 125 µM. Swarming and swimming motilities were inhibited (p < 0.05) by NAC at 250 to 10000 µM. Additionally, 2500 and 10000 µM of NAC reduced biofilm formation. NAC-tobramycin combination showed synergistic effect with FICi of 0.8, and the best combination was 2500-1.07 µM, inhibiting biofilm formation up to 60%, besides reducing pyocyanin and pyoverdine production. Confocal microscopy images revealed a stronger, dense, and compact biofilm of P. aeruginosa PAO1 control, while the biofilm treated with NAC-tobramycin became thinner and more dispersed. Overall, NAC at low concentrations showed promising anti-QS properties against P. aeruginosa PAO1, adding to its already known effect as an antibacterial and antibiofilm agent.

N-acetylcysteine (NAC) attenuates quorum sensing regulated phenotypes in Pseudomonas aeruginosa PAO1

The expression of many virulence genes in bacteria is regulated by quorum sensing (QS), and the inhibition of this mechanism has been intensely investigated. N-acetylcysteine (NAC) has good antibacterial activity and is able to interfere with biofilm-related respiratory infections, but little is known whether this compound has an effect on bacterial QS communication. This work aimed to evaluate the potential of NAC as a QS inhibitor (QSI) in Pseudomonas aeruginosa PAO1 through in silico and in vitro analyses, as well as in combination with the antibiotic tobramycin. Initially, a molecular docking analysis was performed between the QS regulatory proteins, LasR and RhlR, of P. aeruginosa with NAC, 3-oxo-C12-HSL, C4-HSL, and furanone C30. The NAC sub-inhibitory concentration was determined by growth curves. Then, we performed in vitro tests using the QS reporter strains P. aeruginosa lasB-gfp and rhlA-gfp, as well as the expression of QS-related phenotypes. Finally, the synergistic effect of NAC with the antibiotic tobramycin was calculated by fractional inhibitory concentrations index (FICi) and investigated against bacterial growth, pigment production, and biofilm formation. In the molecular docking study, NAC bound to LasR and RhlR proteins in a similar manner to the AHL cognate, suggesting that it may be able to bind to QS receptor proteins in vivo. In the biosensor assay, the GFP signal was turned down in the presence of NAC at 1000, 500, 250, and 125 μM for lasB-gfp and rhlA-gfp (p < 0.05), suggesting a QS inhibitory effect. Pyocyanin and rhamnolipids decreased (p < 0.05) up to 34 and 37%, respectively, in the presence of NAC at 125 μM. Swarming and swimming motilities were inhibited (p < 0.05) by NAC at 250 to 10000 μM. Additionally, 2500 and 10000 μM of NAC reduced biofilm formation. NAC-tobramycin combination showed synergistic effect with FICi of 0.8, and the best combination was 2500–1.07 μM, inhibiting biofilm formation up to 60%, besides reducing pyocyanin and pyoverdine production. Confocal microscopy images revealed a stronger, dense, and compact biofilm of P. aeruginosa PAO1 control, while the biofilm treated with NAC-tobramycin became thinner and more dispersed. Overall, NAC at low concentrations showed promising anti-QS properties against P. aeruginosa PAO1, adding to its already known effect as an antibacterial and antibiofilm agent.

Pseudomonas aeruginosa LasR overexpression leads to a RsaL-independent pyocyanin production inhibition in a low phosphate condition.

Several Pseudomonas aeruginosa virulence-related traits like pyocyanin are regulated by an intricate regulatory network called quorum sensing (QS) that relies on transcriptional regulators that are activated through binding to a self-produced molecule called an autoinducer (AI). QS is composed of three systems, Las, Rhl and Pqs. In the Las system, the regulatory protein LasR interacts with its AI to activate the other two QS systems. In turn, the Rhl and Pqs systems regulate the expression of multiple virulence-related genes, such as the genes of the reiterated operons phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2 involved in pyocyanin production. The Las system also regulates the negative regulator RsaL, which provides negative feedback to the QS-response, including repression of pyocyanin synthesis genes. In this work, we describe that LasR can act as a negative regulator of phzA1 transcription and hence of pyocyanin production and that this regulation is independent of RsaL activity. This work contributes to the understanding of QS-dependent pyocyanin production and demonstrates a previously uncharacterized role of LasR as a repressor.

PqsR-independent quorum-sensing response of Pseudomonas aeruginosa ATCC 9027 outlier-strain reveals new insights on the PqsE effect on RhlR activity.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an opportunistic pathogen that represents an important health hazard. The quorum-sensing response regulates the expression of several virulence factors and involves three regulons: Las, Rhl, and Pqs. The P. aeruginosa ATCC 9027 strain, which belongs to the genetically diverse PA7 clade, contains a frame-shift mutation in the pqsR gene that encodes a transcriptional activator necessary for pyocyanin (PYO) synthesis in type strains PAO1 and PA14. Here we characterize the PqsE-dependent production of PYO in strain ATCC 9027. We show that this strain expresses pqsE independently of PqsR and in the absence of quinolone production, and that PqsE promotes the RhlR-dependent production of PYO, yet this production is not strictly dependent on PqsE. In addition, we show that in both strains ATCC 9027 and PAO1, PqsE overexpression causes an increased concentration of RhlR and enhances PYO production but does not affect rhamnolipids (RL) production in the same way. These results suggest that PqsE interaction with RhlR preferentially modifies its ability to activate transcription of genes involved in PYO production and provide new evidence about PqsE-dependent RhlR activation, highlighting the variability of the QS response among different P. aeruginosa clades and strains. HIGHLIGHTS: Pseudomonas aeruginosa ATCC 9027 is able to produce pyocyanin in phosphate limiting conditions, even in the absence of a functional PqsR. This strain does not produce alkyl quinolones like PQS and HHQ, but expresses pqsE. Synthesis of pyocyanin by ATCC 9027 is only partially dependent on pqsE. The overexpression of pqsE in the ATCC 9027 and PAO1 strains causes pyocyanin overproduction. The overexpression of pqsE in these strains causes an increased RhlR concentration without affecting rhlR transcription or translation. Rhamnolipids production is not affected to the same extent as pyocyanin by overexpression of pqsE in these strains.

Synthesis and biological evaluation of thiazolidinedione derivatives with high ligand efficiency to P. aeruginosa PhzS.

The thiazolidinone ring is found in compounds that have widespan biology activity and there is mechanism-based evidence that compounds bearing this moiety inhibit P. aeruginosa PhzS (PaPzhS), a key enzyme in the biosynthesis of the virulence factor named pyocyanin. Ten novel thiazolidinone derivatives were synthesised and screened against PaPhzS, using two orthogonal assays. The biological results provided by these and 28 other compounds, whose synthesis had been described, suggest that the dihydroquinazoline ring, found in the previous hit (A- Kd = 18 µM and LE = 0.20), is not required for PaPzhS inhibition, but unsubstituted nitrogen at the thiazolidinone ring is. The molecular simplification approach, pursued in this work, afforded an optimised lead compound (13- 5-(2,4-dimethoxyphenyl)thiazolidine-2,4-dione) with 10-fold improvement in affinity (Kd= 1.68 µM) and more than 100% increase in LE (0.45), which follows the same inhibition mode as the original hit compound (competitive to NADH).Executive summaryPhzS is a key enzyme in the pyocyanin biosynthesis pathway in P. aeruginosa.Orthogonal assays (TSA and FITC) show that fragment-like thiazolidinedione derivatives bind to PaPhzS with one-digit micromolar affinity.Fragment-like thiazolidinedione derivatives bind to the cofactor (NADH) binding site in PaPhzS.The molecular simplification optimised the ligand efficiency and affinity of the lead compound.

Lemon Oils Attenuate the Pathogenicity of Pseudomonas aeruginosa by Quorum Sensing Inhibition.

The chemical composition of three Citrus limon oils: lemon essential oil (LEO), lemon terpenes (LT) and lemon essence (LE), and their influence in the virulence factors production and motility (swarming and swimming) of two Pseudomonas aeruginosa strains (ATCC 27853 and a multidrug-resistant HT5) were investigated. The main compound, limonene, was also tested in biological assays. Eighty-four compounds, accounting for a relative peak area of 99.23%, 98.58% and 99.64%, were identified by GC/MS. Limonene (59-60%), γ-terpinene (10-11%) and ß-pinene (7-15%) were the main compounds. All lemon oils inhibited specific biofilm production and bacterial metabolic activities into biofilm in a dose-dependent manner (20-65%, in the range of 0.1-4 mg mL-1) of both strains. Besides, all samples inhibited about 50% of the elastase activity at 0.1 mg mL-1. Pyocyanin biosynthesis decreases until 64% (0.1-4 mg mL-1) for both strains. Swarming motility of P. aeruginosa ATCC 27853 was completely inhibited by 2 mg mL-1 of lemon oils. Furthermore, a decrease (29-55%, 0.1-4 mg mL-1) in the synthesis of Quorum sensing (QS) signals was observed. The oils showed higher biological activities than limonene. Hence, their ability to control the biofilm of P. aeruginosa and reduce the production of virulence factors regulated by QS makes lemon oils good candidates to be applied as preservatives in the food processing industry.

Metabolic engineering of E. coli for pyocyanin production.

Pyocyanin is a secondary metabolite from Pseudomonas aeruginosa that belongs to the class of phenazines, which are aromatic nitrogenous compounds with numerous biological functions. Besides its antifungal and antimicrobial activities, pyocyanin is a remarkable redox-active molecule with potential applications ranging from the pharma industry to the development of microbial fuel cells. Nevertheless, pyocyanin production has been restricted to P. aeruginosa strains, limiting its practical applicability. In this study, the pyocyanin biosynthetic pathway was engineered for the first time for high level production of this compound in a heterologous host. Escherichia coli cells harboring the nine-gene pathway divided into two plasmids were able to produce and secrete pyocyanin at higher levels than some Pseudomonas aeruginosa strains. The influence of culture and induction parameters were evaluated, and the optimized conditions led to an increase of 3.5-fold on pyocyanin accumulation. Pathway balancing was achieved by testing a set of plasmids with different copy numbers to optimize the expression levels of pyocyanin biosynthetic genes, resulting in a fourfold difference in product titer among the engineered strains. Further improvements were achieved by co-expression of Vitreoscilla hemoglobin Vhb, which relieved oxygen limitations and led to a final titer of 18.8 mg/L pyocyanin. These results show promise to use E. coli for phenazines production, and the engineered strain developed here has the potential to be used in electro-fermentation systems where pyocyanin plays a role as electron-shuttle.

Optimized Production of a Redox Metabolite (pyocyanin) by Pseudomonas aeruginosa NEJ01R Using a Maize By-Product.

Pseudomonas aeruginosa metabolizes pyocyanin, a redox molecule related to diverse biological activities. Culture conditions for the production of pyocyanin in a defined medium were optimized using a statistical design and response surface methodology. The obtained conditions were replicated using as substrate an alkaline residual liquid of cooked maize and its by-products. The untreated effluent (raw nejayote, RN) was processed to obtain a fraction without insoluble solids (clarified fraction, CL), then separated by a 30 kDa membrane where two fractions, namely, retentate (RE) and filtered (FI), were obtained. Optimal conditions in the defined medium were 29.6 °C, 223.7 rpm and pH = 6.92, which produced 2.21 µg mL-1 of pyocyanin, and by using the wastewater, it was possible to obtain 3.25 µg mL-1 of pyocyanin in the retentate fraction at 40 h. The retentate fraction presented the highest concentration of total solids related to the maximum concentration of pyocyanin (PYO) obtained. The pyocyanin redox behavior was analyzed using electrochemical techniques. In this way, valorization of lime-cooked maize wastewater (nejayote) used as a substrate was demonstrated in the production of a value-added compound, such as pyocyanin, a redox metabolite of Pseudomonas aeruginosa NEJ01R.

Human probiotic bacteria attenuate Pseudomonas aeruginosa biofilm and virulence by quorum-sensing inhibition.

This work investigated chloroform extracts from culture supernatants of two human probiotic bacteria, Lactobacillus casei CRL 431 and Lactobacillus acidophilus CRL 730 for the production of virulence factors and quorum sensing (QS) interference against three Pseudomonas aeruginosa strains. Both extracts inhibited biofilm biomass (up to 50%), biofilm metabolic activity (up to 39%), the production of the enzyme elastase (up to 63%) and pyocyanin (up to 77%), and decreased QS, without presenting any antibacterial acgivity. In addition, the chloroform extracts of both strains disrupted preformed biofilms of the three strains of P. aeruginosa analyzed (up to 40%). GC-MS analysis revealed that the major compounds detected in the bioactive extracts were four diketopiperazines. This study suggests that the metabolites of L. casei and L. acidophilus could be a promising alternative to combat the pathogenicity of P. aeruginosa.

Calycopterin, a major flavonoid from Marcetia latifolia, modulates virulence-related traits in Pseudomonas aeruginosa.

Although bacterial resistance is a worldwide growing concern, the development of bacteriostatic and bactericidal drugs has been decreasing in the last decade. Compounds that modulate the microorganism virulence, without killing it, have been considered promising alternatives to combat bacterial infections. However, most signaling pathways that regulate virulence are complex and not completely understood. The rich chemical diversity of natural products offers a good starting point to identify key compounds that shed some light on this matter. Therefore, we investigated the role of Marcetia latifolia ethanolic extract, as well as its major constituent, calycopterin (5,4'-dihydroxy-3,6,7,8-tetramethoxylflavone), in the regulation of virulence-related phenotypes of Pseudomonas aeruginosa. Our results show that calycopterin inhibits pyocyanin production (EC50 = 32 µM), reduces motility and increases biofilm formation in a dose-dependent manner. Such biological profile suggests that calycopterin modulates targets that may act upstream the quorum sensing regulators and points to its utility as a chemical probe to further investigate P. aeruginosa transition from planktonic to sessile lifestyle.

The third quorum-sensing system of Pseudomonas aeruginosa: Pseudomonas quinolone signal and the enigmatic PqsE protein.

Pseudomonas aeruginosa is an opportunistic pathogen that produces several virulence factors such as lectin A, pyocyanin, elastase and rhamnolipids. These compounds are controlled transcriptionally by three quorum-sensing circuits, two based on the synthesis and detection of N-acyl-homoserine-lactone termed the Las and Rhl system and a third system named the Pseudomonas quinolone signal (PQS) system, which is responsible for generating 2-alkyl-4(1 h)-quinolones (AQs). The transcriptional regulator called PqsR binds to the promoter of pqsABCDE in the presence of PQS or HHQ creating a positive feedback-loop. PqsE, encoded in the operon for AQ synthesis, is a crucial protein for pyocyanin production, activating the Rhl system by a still not fully understood mechanism. In turn, the regulation of the PQS system is modulated by Las and Rhl systems, which act positively and negatively, respectively. This review focuses on the PQS system, from its discovery to its role in Pseudomonas pathogenesis, such as iron depletion and pyocyanin synthesis that involves the PqsE protein - an intriguing player of this system.

Structure-based Druggability Assessment of Anti-virulence Targets from Pseudomonas aeruginosa.

Antimicrobial Resistance (AMR) represents a serious threat to health and the global economy. However, interest in antibacterial drug development has decreased substantially in recent decades. Meanwhile, anti-virulence drug development has emerged as an attractive alternative to fight AMR. Although several macromolecular targets have been explored for this goal, their druggability is a vital piece of information that has been overlooked. This review explores this subject by showing how structure- based freely available in silico tools, such as PockDrug and FTMap, might be useful for designing novel inhibitors of the pyocyanin biosynthesis pathway and improving the potency/selectivity of compounds that target the Pseudomonas aeruginosa quorum sensing mechanism. The information provided by hotspot analysis, along with binding site features, reveals novel druggable targets (PhzA and PhzS) that remain largely unexplored. However, it also highlights that in silico druggability prediction tools have several limitations that might be overcome in the near future. Meanwhile, anti-virulence drug targets should be assessed by complementary methods, such as the combined use of FTMap/PockDrug, once the consensus druggability classification reduces the risk of wasting resources on undruggable proteins.

Pyocyanin Restricts Social Cheating in Pseudomonas aeruginosa.

Quorum sensing (QS) in Pseudomonas aeruginosa coordinates the expression of virulence factors, such as exoproteases and siderophores, that are public goods utilized by the whole population of bacteria, regardless of whether they invested or not in their production. These public goods can be used by QS defective mutants for growth, and since these mutants do not contribute to public goods production, they are considered social cheaters. Pyocyanin is a phenazine that is a toxic, QS-controlled metabolite produced by P. aeruginosa. It is a redox-active compound and promotes the generation of reactive oxygen species; it also possesses antibacterial properties and increases fitness in competition with other bacterial species. Since QS-deficient individuals are less able to tolerate oxidative stress, we hypothesized that the pyocyanin produced by the wild-type population could promote selection of functional QS systems in this bacterium. Here, we demonstrate, using competition experiments and mathematical models, that, indeed, pyocyanin increases the fitness of the cooperative QS-proficient individuals and restricts the appearance of social cheaters. In addition, we also show that pyocyanin is able to select QS in other bacteria such as Acinetobacter baumannii.

Virulence attributes in Brazilian clinical isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen responsible for causing a huge variety of acute and chronic infections with significant levels of morbidity and mortality. Its success as a pathogen comes from its genetic/metabolic plasticity, intrinsic/acquired antimicrobial resistance, capacity to form biofilm and expression of numerous virulence factors. Herein, we have analyzed the genetic variability, antimicrobial susceptibility as well as the production of metallo-ß-lactamases (MBLs) and virulence attributes (elastase, pyocyanin and biofilm) in 96 strains of P. aeruginosa isolated from different anatomical sites of patients attended at Brazilian hospitals. Our results revealed a great genetic variability, in which 86 distinct RAPD types (89.6% of polymorphisms) were detected. Regarding the susceptibility profile, 48 strains (50%) were resistant to the antimicrobials, as follows: 22.92% to the three tested antibiotics, 12.5% to both imipenem and meropenem, 11.46% to ceftazidime only, 2.08% to imipenem only and 1.04% to both ceftazidime and meropenem. Out of the 34 clinical strains of P. aeruginosa resistant to both imipenem and meropenem, 25 (73.53%) were MBL producers by phenotypic method while 12 (35.29%) were PCR positive for the MBL gene SPM-1. All P. aeruginosa strains produced pyocyanin, elastase and biofilm, although in different levels. Some associations were demonstrated among the susceptibility and/or production of these virulence traits with the anatomical site of strain isolation. For instance, almost all strains isolated from urine (85.71%) were resistant to the three antibiotics, while the vast majority of strains isolated from rectum (95%) and mouth (66.67%) were susceptible to all tested antibiotics. Urine isolates produced the highest pyocyanin concentration (20.15±5.65 µg/ml), while strains isolated from pleural secretion and mouth produced elevated elastase activity (1441.43±303.08 FAU) and biofilm formation (OD590 0.676±0.32), respectively. Also, MBL-positive strains produced robust biofilm compared to MBL-negative strains. Collectively, the production of site-dependent virulence factors can be highlighted as potential therapeutic targets for the treatment of infections caused by heterogeneous and resistant strains of P. aeruginosa.